Biological activities of cystic fibrosis serum III. CF serum induced uptake of 45Ca++ by rabbit tracheal explants

Vol. 88, No. 4, 1979

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

June 27, 1979

Pages 1398-l

404

BIOLOGICALACTIVITIES OF CYSTICFIBROSIS SERUM III.

CF SERUMINDUCEDUPTAKEOF 45CAu BY RABBIT TRACHEALEXPLANTS

BRUCEIAN BOGART NewYork University School of Medicine Department of Cell Biology, NewYork, NY 10016 ELAINE J. CONOD,PUERZAF. GAERLAN,CAROLYN R. DENNING,JAMESCONOVER St. Vincent's Hospital and Medical Center of New York and NewYork University School of Medicine, NewYork, NY 10016 Received

May 21,

1979

SUMMARY. Cystic Fibrosis (CF) serum and its isolated component IgG fraction produce an Increased uptake of 45Ca++ in rabbit tracheal explants when compared to control serum and it isolated IgG fraction. Heterozygote serum also produced sn increased uptake of &%a++ but not to the sameextent as CF serum. The calcium channel blocker D600 inhibited the CF serum induced uptake of ?%a++ indicating that CF serum may be acting on the plasma membraneto produce changes in calcium permeability in rabbit tracheal explants. INTBCDUCTION. Cystic fibrosis

(CF) is a genetic disease that is characterized

of the exocrine glands. including

Clinically,

the patients present a triad

chronic pulmonary disease, exocrine pancreatic

elevated levels of sweat sodium and chloride. heterozygote

individuals

The CF serum factor(s)

mucoussecretion in rabbit (3,4).

Our laboratory

induced ciliary

tracheal explants (1,2,3)

(5,6) has investigated

dyskinesia reaction

in rabbit

calcium dependence in the CF serum ciliary experimentally

in their

has been observed to produce ciliary

increasing intracellular

of symptoms,

insufficiencies

In addition,

have an abnormal factor(s)

by dysfunction

CF patients

and and

serum (l,2,3). dyskinesia and

and oyster gill

preparations

the role of calcium in the CF serum tracheal explants.

Evidence for a

dyskinesia reaction has been obtained by levels of calcium with the calcium ionophore

A23187 or reducing the calcium concentration

of serum and/or mediumby the addition

of the calcium chelator EGTA. During experiments designed to increase intracellular calcium, we observed changes in ciliary

function,

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mucous secretion and changes in

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autophagic vacuoles similar

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to those observed after

rabbit

tracheal

explants were

incubated with CF serum. Addition of the calcium chelator EGTA effectively the ciliary

dyskinesia response to CF serum or to the calcium ionophore A23187 in

rabbit tracheal

explants.

serum-like ciliary trations

equivalent

In addition,

the calcium ionophore A23187 produced a CF

dyskinesia reaction only if the mediumcontained calcium concento serum.

This communication describes a quantitative

in 45ca++ uptake induced by serum from CF and heterozygous individuals fraction

inhibited

derived from CF serum in rabbit

tracheal explants.

channel blocker methoxyverapamil inhibits

increase

or by IgG

In addition,

the calcium

the 45Ca++uptake induced by CF serum.

MATERIALSANDMETHODS. After informed consent (12), venous blood is drawn and serum prepared as previously described (2,5), while, rabbit tracheal explants are prepared weekly (2,5,6). Tracheal explants are prepared under conditions identical to the bioassay (2,5) and incubated with 1 ml of either control, heterozygote or CF serum or fractions in phosphate buffer, in multiwell plastic culture dishes. Five pCi/ml i? Ca++ and 5 pCi/ml 3H inulin (Specific activity of 301 mCi/g) are added. The latter is used as a marker for extracellular space. The explants are removed from the serum at 0 time and 3 and 6 minute intervals, washed and blotted, weighed and dried overnight at 100' C. After cooling to room temperature, the explants are weighed and dissolved in 1.5 ml Protosol tissue solubilizer (New England Nuclear) at 50' C in vials fitted with teflon lined caps. After cooling, 0.5 ml of glacial acetic acid is added to neutralize the alkaline solution and reduce the chemiluminescence. This procedure is followed by the addition of 15 ml of Aquasol liquid scintillation solution (New England Nuclear) and counted on a Beckmanliquid scintillation spectrometer. After correction for background and the inulin space, the uptake of 45Ca++ into the calcium space of the rabbit tracheal explants is calculated as the ratio of counts per minute per milligram tissue/counts per minute per milliliter medium (7). DEAE-cellulose separated serum IgG fractions (IgG) is prepared as previously described (8). Statistical analysis is performed by Student's t test. RESULTSANDDISCUSSION. CF serum induced a significantly explant than did control ciliary

greater uptake of 45,,++

serum during the time intervals

dyskinesia bioassay ( P < 0.1).

by the rabbit tracheal

that coincided with the

Indeed, the uptake of 45Ca++ induced by CF

serum at 3 minutes was greater that the 45,,++ uptake induced by control serum after either

3 minutes or 6 minutes (figure

1).

Heterozygote serum induced a 45C,++ uptake

which was intermediate between the values produced by CF and control There was an overlap in the values for 45,,++

uptake produced by heterozygote

and CF serum, while both values were significant values for 45Ca++uptake produced by control

1399

serum (figure

(P

1)

serum

< 0.1) when comparedwith the

serum (figure

1).

Addition of 1 mMD600

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3

Time Figure 1.

6

in Minutes

Uptake of "Ca++ into . calcium space in rabbit incubated in serum drawn from control cystic

fibrosis

(a)

individuals.

tracheal explant

(@), heterozygote

(A)

and

The calcium space is calcu-

lated as cpm '*Ca++ per mg tissue/cpm '*,a*

per mg media with

cpmper mg tissue corrected for the b -inulin and CF sera and 6 for heterozygote

sera).

space (N=7 for control

Data represents the mean

-+ S.E.M. for each set of data.

to aliquots of CF serum samples produced a significant

inhibition

uptake induced by CF serum in the rabbit tracheal explant The IgC fraction cellulose

(figure

derived from the serum of CF individuals

has been implicated in both ciliary

rabbit tracheal bioassay (8,9,10).

2).

separated on DFAE

dyskinesia and mucous secretion in the

The IgC fractions

1400

of the 45ca++

(1 m&ml) derived from the

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AND

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3

Dptake of 45Ca++ into

2.

plants

CF serum plus (m).

(P < 0.01, buffer

derived

see figure

produced

3).

a significant

but not at concentrations These results increase

indicate

in 45Ca++ uptake

of the ciliary

the addition

produced

dyskinesia

space of rabbit

of CF serum (A),

a significant

from control

uptake

of 45,,++

or with

of 10 pg/ml or 1 pg/ml (figure CF serum or IgG fractions

in the rabbit bioassay.

tracheal

As stated

1401

explants

of D600

when compared with buffer

diluted

with

IgG fractions

ex-

the mean + S.E.M.

phosphate

45ca++ uptake at concentrations

that

antagonist

Data represents

individuals

The CF serum derived

tracheal

or in the presence

of 1 mM of the calcium

(N = 6 sera from patients).

serum of CF individuals the IgG fractions

in Minutes

the calcium

in the presence

COMMUNICATIONS

6

Time Figure

RESEARCH

alone phosphate

of 1 mg/ml or 100 pg/ml

4). from CF serum produced during

an

the time intervals

above, the uptake of 45Ca++ was greater

Vol. 88, No. 4, 1979

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6

3

Time in Minutes Uptake of 45ca++ into the calcium space of rabbit

Figure 3.

tracheal

explants in the presence of 1 mg/ml serum IgG fractions from control buffer after

(A).

(0) and CF Fndividuals

m)

derived

and phosphate elution

Data represents the mean+ S.E.M.

3 minutes of incubation in CF serum than the 45Ca++uptake produced by control

serum at either correlated

3 minutes or 6 minutes of incubation.

with the onset of the ciliary

observed after

This observation can be

dyskinesia reaction

(2) which often was

3 minutes of incubation with CF serum and also with the observation

that control serum does not induce the ciliary produced the ciliary

dyskinesia reaction

dyskinesia reaction.

CF serum often

in rabbit tracheal explants after

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3 minutes

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Dose dependent uptake of 45,,++ induced by CF serum derived IgG

Figure 4.

fractions

after

induce different

6 minutes.

Different

concentrations

of CF IgG

uptake of 45,,++ into the calcium space.

response is compared with 45Ca++uptake induced by control CF serum and phosphate buffer

after

and almost always,af%er 6 minutes, while control

trigger

serum aid not produce this reaction. that there may be a

level of calcium uptake by epithelium of the rabbit the ciliary

uptake after

serum,

6 minutes.

These results along with cumulative evidence (2,5) indicate critical

This

tracheal explant to

dyskinesia reaction and that CF serum can produce this level of

only 3 minutes, while control serum does not induce the necessary

calcium uptake by the rabbit The ~600 inhibition

tracheal

explant even after

6 minutes.

of the 45ca++ uptake induced by CF serum is also indicative

of a calcium dependence in this reaction.

Wehave previously

can inhibit

(5) by reducing the calcium concentration

the ciliary

dyskinesia reaction

1403

demonstrated that EGTA

Vol. 88, No. 4, 1979

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

in serum or in mediumcontaining the calcium ionophore A23187. D600 is thought to exert its

effect

on cells by preventing

level of the calcium channels (11). 45Ca++ in rabbit further

indicates

Ca++ influx

The inhibition

tracheal explants by a specific

across the cell membraneat the of the stimulated uptake of

calcium channel blocker (D600)

that CF serum alters membranepermeability

to 45Ca++.

These experimental findings in conjunction with our previous observations (5,6,10) rabbit

indicate

a calcium dependencein the ciliary

tracheal explant bioassay system and further

factor(s) the ciliary

dyskinesia reaction indicates

in the

that the CF serum

may be acting at the level of the plasma membraneduring the stimulation dyskinesia reaction in rabbit

Recently, Banschbach et al.

of

tracheal explants.

(12) have also reported that a CF serum promoted an

increase in 45Ca++ labeling in normal humanleukocytes of a similar magnitude as observed in this study, indicating uptake in several different

that CF serum can produce an increase in Ca*

cell types.

Acknowledgement. The authors wish to thank Mr. Chrisopher Keegan for his expert technical assistance and Ms. Susan Blond for typing the manuscript. This work was supported in part by Cystic Fibrosis Foundation and National Foundation of March of Dimes. REFERENCES.

1. 2.

3. 4. 2: 7. 8. 9. 10. 11. 12.

13.

Speck, A., Heich, H.M.C., Cress, H. and Logan, W.S. Pediatr.

Res. 1:173-177.

1967.

Conover, J.H., Bonforte, F.J., Hathway, P., Padiuc, D., Conod, E.J., Hirschhorn, K. and Kopel, F.B. Pediatr. Res. 1:220-223. 1973. diSant!Agnese, P.A. and David, P.A. N. Eng. J. Med. 295:481-485,543-541,597-602.

1977.

Bowman,B.H., Lockhart, L.H. and McCombs,M.L. Science. 164:325-326. 1969. 1977. Bogart, B.I., Conod, E.J. and Conover, J.H. Pediatr. Res. 2:131-134. Bogart, B.I., Conod, E.J., Gaerlan, P.F. and Conover, J.H. Pediatr. Res. -12:15-24.

1978.

Putney, J.W. J. Pharmacut. and Expt. Therap. 199:526-537. 1976. Conod, E., Gaerlan, P., and Conover, J.H. Pediatr. Res. g:45-47. 1977. Bowman,B., Lankford, B., Fuller, G., Carson, S., Kurosky, A. and Barnett, K. Biochem. Biophys. Res. Commun.64:1310-1315. 1975. Conover, J.H. and Conod, E.J. Biochem. Biophys. Res. Commun.83:1595-1601. 1978. Fleckenstein, A., Nakayama, K., Fleckenstein-Grun, G. and Byon, Y.X. In: Calcium transport in contraction and secretion. Carafoli, E., Clementi, E., Drabikowski, W. and Margreth, A. Eds. p. 555-566. North-Holland, Amsterdam. 1975. Banschbach, M.W., Karam, A.G., Love, P.K. and Hilman, B.C. Biochem. Biophys. Res. COD. 84: 922. 1978. Serum sampleswere obtained under the protocols approved by the Research Associate: Committees of St. Vincent's Hospital and Medical Center and NewYork University School of Medicine

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